SUMMARY HIV-1 infection requires a life-term antiretroviral treatment because its cessation leads to rapid viral rebound from the HIV-1 latent cellular/tissue reservoir. Novel approaches to eradicate or permanently silence HIV-1 proviruses are urgently needed to achieve a ?sterile? cure of HIV infection, for which CRIPSR/Cas9 genome editing has opened a new avenue. In the past years, we and others have utilized Cas9-mediated genome editing to excise HIV-1 provirus in vitro, ex vivo and in vivo. One of challenges before clinical application is how to deliver effectively, specifically and safely the powerful genome editing machinery to HIV-1 latently infected cells. The objective of this proposal is to develop novel synthetic nanoparticle (NP) for the in vivo delivery of Cas9/sgRNA ribonucleoprotein (RNP) specifically to CD4 T cells, the most important HIV-1 latent cellular reservoir. We have recently developed a novel synthetic PEG-Morpholine copolymer (PEG-pMor) NP system for in vivo drug delivery in mouse model. Our preliminary data demonstrated the feasibility and efficiency of this PEG-pMor NP to deliver Cas9/sgRNA plasmid or RNP in multiple organs/tissues resulting in eradication of HIV-1 proviral DNA or host cellular genes in vivo. In this proposal, a novel CD4-specific designed ankyrin repeat protein (DARPin) peptide will be displayed on the surface of the PEG-pMor NP to achieve targeted delivery of Cas9/sgRNA RNP to human CD4 T cells in HIV-1-infected humanized mouse models. In Aim I, we will develop and characterize CD4 T cell-targeting NP both in vitro and in vivo and determine the efficiency of CD4-specific DARPin-mediated Cas9/duplex sgRNA RNP to excise CCR5 gene in human primary T cells (in vitro) and humanized mouse model (in vivo). In Aim II, we will determine the efficiency of CD4 T cell- targeting NP for in vivo delivery of Cas9/sgRNA RNP to excise HIV-1 proviral DNA. In Aim III, we will assess the combinatory therapeutic potential of CD4 T cell-targeting NP in vivo delivery of Cas9/quadruplex sgRNA RNP (LTR1/GagD+CCR5-A/B) in blocking or delaying latent HIV-1 viral rebound in humanized mouse model. This high-reward proposal focuses on the screening of novel CD4 T cell-specific delivery of RNP genome editors to excise HIV-1 provirus and disable HIV-1 entry coreceptor CCR5 gene. The positive outcome will offer a novel tool to deliver Cas9/sgRNA RNP to CD4 T cells and/or other reservoir cells in vivo, and thus provide new avenues for the development of therapeutics to cure HIV-1.